Cytosine methylation in DNA is an epigenetic mechanism regulating gene expression and plays a vital role in cell differentiation or proliferation. Tumor cells often exhibit aberrant DNA methylation, e.g. hypermethylation of tumor suppressor gene promoters. New methods, capable of determining methylation status of specific DNA sequences, are thus being developed. Among them, MS-HRM (methylation-specific high resolution melting) and electrochemistry offer relatively inexpensive instrumentation, fast assay times and possibility of screening multiple samples/DNA regions simultaneously. MS-HRM is due to its sensitivity and simplicity an interesting alternative to already established techniques, including methylation-specific PCR or bisulfite sequencing. Electrochemistry, when combined with suitable electroactive labels and electrode surfaces, has been applied in several unique strategies for discrimination of cytosines and methylcytosines. Both techniques were successfully tested in analysis of DNA methylation within promoters of important tumor suppressor genes and could thus help in achieving more precise diagnostics and prognostics of cancer. Aberrant methylation of promoters has already been described in hundreds of genes associated with tumorigenesis and could serve as important biomarker if new methods applicable into clinical practice are sufficiently advanced.Key words: DNA methylation - 5-methylcytosine - HRM analysis - melting temperature - DNA duplex - electrochemistry - nucleic acid hybridizationThis work was supported by MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 16. 5. 2016.

• MeSH
• 5-methylcytosin analýza   MeSH
• cytosin analýza   MeSH
• denaturace nukleových kyselin MeSH
• DNA analýza   MeSH
• elektrochemie metody   MeSH
• lidé MeSH
• metylace DNA * MeSH
• tumor supresorové geny * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• 5-methylcytosin MeSH
• cytosin MeSH
• DNA MeSH

Here, we present a practical overview of four commonly used validation methods for DNA methylation assessment: methylation specific restriction endonucleases (MSRE) analysis, pyrosequencing, methylation specific high-resolution DNA melting (MS-HRM) and quantitative methylation specific polymerase chain reaction (qMSP). Using these methods, we measured DNA methylation levels of three loci in human genome among which one was highly methylated, one intermediately methylated and one unmethylated. We compared the methods in terms of primer design demands, methods' feasibility, accuracy, time and money consumption, and usability for clinical diagnostics. Pyrosequencing and MS-HRM proved to be the most convenient methods. Using pyrosequencing, it is possible to analyze every CpG in a chosen region. The price of the instrument may represent the main limitation of this methodology. MS-HRM is a simple PCR-based method. The measurement was quick, cheap and very accurate. MSRE analysis is based on a methylation specific digestion of DNA. It does not require a bisulfite conversion of DNA as the other methods. MSRE analysis was very easy to perform, however, it was not suitable for intermediately methylated regions and it was also quite expensive. qMSP is a qPCR-based method that uses primers designed specifically for methylated and unmethylated alleles of a chosen region. This was the least accurate method and also the primer design and optimization of PCR conditions were highly demanding.

• Klíčová slova
• DNA methylation, MS-HRM, MSRE, Pyrosequencing, Validation methods, qMSP,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Rilmenidine is an alpha 2 adrenoreceptor agonist used in the treatment of mild and moderate hypertension. In this study, a fast and accurate liquid chromatographic method with tandem mass spectrometric detection has been validated in order to assure quantification of rilmenidine in human serum. The fragmentation pathway of protonated rilmenidine was studied using high-resolution mass spectrometry (HRMS). This study compared selectivity, linearity, accuracy, precision, extraction efficiency, matrix effect and sensitivity using common liquid-liquid extraction (LLE) and solid-phase extraction (SPE) procedures. The limit of quantitation for both extraction techniques was 0.1 ng/ml. Several differences between the LLE and SPE have been observed in terms of linearity, accuracy, precision and matrix effect. Additionally, the advantages of SPE included less manual work load and increased recovery of rilmenidine in human serum to approximately 80% (LLE, 57%). The developed method involving SPE was found to be accurate (relative error (RE) < 5%), reproducible (relative standard deviation, RSD < 7%), robust and suitable for quantitative analysis of rilmenidine in serum samples obtained from patients under antihypertensive treatment.

• MeSH
• antihypertenziva krev   chemie   farmakokinetika   MeSH
• chromatografie kapalinová metody   MeSH
• extrakce na pevné fázi MeSH
• lidé MeSH
• metoda nejmenších čtverců MeSH
• oxazoly krev   chemie   farmakokinetika   MeSH
• reprodukovatelnost výsledků MeSH
• rilmenidin MeSH
• senzitivita a specificita MeSH
• stabilita léku MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• antihypertenziva MeSH
• oxazoly MeSH
• rilmenidin MeSH

Popularity of natural-based preparations supporting the sexual potency significantly increased in recent years, which also led to the increase of illegal use of phosphodiesterase type 5 inhibitor (PDE-5) in sexual performance enhancement products. In this study, a rapid U-HPLC‒HRMS/MS method has been developed to simultaneously determine 59 PDE-5 inhibitors and their analogues. Within the development of sensitive method for analysis of 59 PDE-5 inhibitors and their analogues, both sample preparation procedure, as well as separation / detection conditions have been optimized. Extraction efficiency of particular extraction solvents, influence of different mobile phase additives on target analytes separation, as well as impact of various settings of mass analyzer on sensitivity of detection were examined. Data were collected in the 'full MS/data dependent MS/MS' acquisition mode (full MS-dd-MS/MS). Before the U-HPLC‒HRMS/MS method was used for analysis of real samples, proper validation had been conducted. The precision of the method expressed as the relative standard deviation (RSD) was ≤4.2% and ≤5.2% at spiking concentrations 5 μg/g and 0.25 μg/g, respectively. The limits of quantification were in the range 0.25 - 0.05 μg/g and the recovery ranged between 71 and 90%. The optimized method was successfully applied for analysis of 64 real samples, and 10 of them were proved to contain both registered or unregistered synthetic PDE-5 inhibitors. Additionally, the acquired U-HPLC‒HRMS/MS fingerprints were demonstrated to serve as an efficient tool for revealing of other type of possible fraud in products labeling. Retrospective mining of markers of herbs declared on dietary supplements packaging allowed to assess the trueness / untruth in the declaration of medical herbs composition.

• Klíčová slova
• PDE-5 inhibitors, adulteration, dietary supplements, high resolution tandem mass spectrometry, orbitrap,
• MeSH
• fytonutrienty analýza   normy   MeSH
• inhibitory fosfodiesterasy 5 analýza   MeSH
• kontaminace léku prevence a kontrola   MeSH
• limita detekce MeSH
• padělané léky analýza   MeSH
• podvod prevence a kontrola   MeSH
• potravní doplňky analýza   normy   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• fytonutrienty MeSH
• inhibitory fosfodiesterasy 5 MeSH
• padělané léky MeSH

In this series of two papers, 192 doping agents belonging to the classes of stimulants, narcotics, cannabinoids, diuretics, β2-agonists, β-blockers, anabolic agents, and hormone and metabolic modulators were investigated, with the aim to assess the benefits and limitations of ion mobility spectrometry (IMS) in combination with ultra-high performance liquid chromatography (UHPLC) and high resolution mass spectrometry (HRMS) in anti-doping analysis. In this first part, a generic UHPLC-IM-HRMS method was successfully developed to analyze these 192 doping agents in standard solutions and urine samples, and an exhaustive database including retention times, TWCCSN2 values, and m/z ratios was constructed. Urine samples were analyzed using either a simple "dilute and shoot" procedure or a supported liquid-liquid extraction (SLE) procedure, depending on the physicochemical properties of the compounds and sensitivity criteria established by the World Anti-Doping Agency (WADA) as the minimum required performance levels (MRPL). Then, the precision of the generic UHPLC-IM-HRMS method was assessed as intraday, interday as well as interweek variation of UHPLC retention times and TWCCSN2 values, for which RSD the values were always lower than 2% in urine samples. The possibility to filter MS data using IMS dimension was also investigated, and in average, the application of IMS filtration provided low energy MS spectra with 86% less interfering peaks in both standard and urine samples. Therefore, the filtered MS spectra allowed for an easier interpretation and a lower risk of false positive result interpretations. Finally, IMS also offers additional selectivity to the UHPLC-HRMS enabling to separate isobaric and isomeric substances. Among the selected set of 192 doping agents, there were 30 pairs of isobaric or isomeric compounds, and only two pairs could not be resolved under the developed conditions. This illustrates the potential of adding ion mobility to UHPLC-HRMS in anti-doping analyses.

• Klíčová slova
• Collision cross section, Doping analysis, High resolution mass spectrometry, Ion mobility spectrometry, Ultra-high performance liquid chromatography,
• MeSH
• anabolika * MeSH
• doping ve sportu * MeSH
• hmotnostní spektrometrie MeSH
• iontová mobilní spektrometrie MeSH
• odhalování abúzu drog MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• anabolika * MeSH

The objective of this study was to develop and validate a 96-well plate solid phase extraction method for analysis of 23 lipophilic persistent organic pollutants (POPs) in low-volume plasma and serum samples which is applicable for biomonitoring and epidemiological studies. The analysis of selected markers for internal exposure: 16 polychlorinated biphenyls (PCBs), 5 organochlorine pesticides (OCPs), octachlorinated dibenzo-p-dioxin (OCDD), and polybrominated diphenylether 47 (BDE 47) was evaluated by comparing two SPE sorbents and GC-HRMS or GC-MS/MS detection. The final method extracted 23 POPs from 150 μL of serum and plasma using a 96-well extraction plate containing 60 mg Oasis HLB sorbent per well prior to GC-HRMS magnetic sector analysis. The extraction method was applied to 40 plasma samples collected for an epidemiological study. The recovery of selected POPs ranged from 31% to 63% (n = 48), and detection limits ranged from 2.2 to 45 pg/mL for PCBs, 4.2 to 167 pg/mL for OCPs, 7.8 pg/mL for OCDD and 6.1 pg/mL for BDE 47. This method showed good precision with relative standard deviations of selected POP concentrations in quality control samples (n = 48) ranging from 11% to 25%. The trueness was determined with standard reference material serum (n = 48) and the deviation from certified values ranged from 1 to 27%. Of the 23 POPs analyzed, 18 were detected in 43% to 100% of plasma samples collected for the epidemiological study. The method showed good robustness with low inter-well plate variation (11-31%) determined by twelve 96-well plate extractions, and can extract 96 samples, including quality controls and procedural blanks in 2-3 days. Comparison with GC-MS/MS analysis showed that similar concentrations (within 0.5% to 30%) of most POPs could be obtained with GC-APCI-MS/MS. Larger deviations were observed for PCB 194 (60%) and trans-nonachlor (43%). The developed method produces accurate concentrations of low-level marker POPs in plasma and serum, providing a suitable high-throughput sample preparation procedure for biomonitoring and epidemiological studies involving large sample size and limited sample volume. GC-HRMS was chosen over GC-MS/MS, however the latter showed promising results, and could be used as an alternative to GC-HRMS analysis for most POPs.

• Klíčová slova
• APCI-MS/MS, GC-HRMS, High-throughput SPE, Persistent organic pollutants,
• MeSH
• extrakce na pevné fázi přístrojové vybavení   metody   MeSH
• látky znečišťující životní prostředí krev   MeSH
• lidé MeSH
• limita detekce MeSH
• monitorování životního prostředí metody   MeSH
• organické látky krev   MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
• reprodukovatelnost výsledků MeSH
• tandemová hmotnostní spektrometrie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• látky znečišťující životní prostředí MeSH
• organické látky MeSH

RATIONALE: On-line chemical characterization methods of atmospheric aerosols are essential to increase our understanding of physicochemical processes in the atmosphere, and to study biosphere-atmosphere interactions. Several techniques, including aerosol mass spectrometry, are nowadays available, but they all suffer from some disadvantages. In this research, desorption atmospheric pressure photoionization high-resolution (Orbitrap) mass spectrometry (DAPPI-HRMS) is introduced as a complementary technique for the fast analysis of aerosol chemical composition without the need for sample preparation. METHODS: Atmospheric aerosols from city air were collected on a filter, desorbed in a DAPPI source with a hot stream of toluene and nitrogen, and ionized using a vacuum ultraviolet lamp at atmospheric pressure. To study the applicability of the technique for ambient aerosol analysis, several samples were collected onto filters and analyzed, with the focus being on selected organic acids. To compare the DAPPI-HRMS data with results obtained by an established method, each filter sample was divided into two equal parts, and the second half of the filter was extracted and analyzed by liquid chromatography/mass spectrometry (LC/MS). RESULTS: The DAPPI results agreed with the measured aerosol particle number. In addition to the targeted acids, the LC/MS and DAPPI-HRMS methods were found to detect different compounds, thus providing complementary information about the aerosol samples. CONCLUSIONS: DAPPI-HRMS showed several important oxidation products of terpenes, and numerous compounds were tentatively identified. Thanks to the soft ionization, high mass resolution, fast analysis, simplicity and on-line applicability, the proposed methodology has high potential in the field of atmospheric research.

• MeSH
• aerosoly analýza   chemie   MeSH
• atmosféra MeSH
• chromatografie kapalinová metody   MeSH
• hmotnostní spektrometrie přístrojové vybavení   metody   MeSH
• kapryláty analýza   chemie   MeSH
• kyseliny karboxylové analýza   chemie   MeSH
• oxidace-redukce MeSH
• terpeny analýza   chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aerosoly MeSH
• kapryláty MeSH
• kyseliny karboxylové MeSH
• octanoic acid MeSH Prohlížeč
• terpeny MeSH

There is an increased need for quick screening tools enabling the detection of Waste Electrical and Electronic Equipment (WEEE), and in particular brominated flame retardants (BFRs), in polymeric materials. Unfortunately, common laboratory techniques might face matrix effects or encounter long sample preparation times. Therefore, an ambient desorption mass spectrometric technique such as Direct Analysis in Real Time - High Resolution Mass Spectrometry (DART-HRMS) might provide fast BFR identification in polymeric objects. Within this pilot-study, the potential of DART-HRMS for the detection of WEEE fractions has been tested on WEEE impacted consumer goods such as toys and food contact articles. The identification of polymeric material containing WEEE to date has relied on measuring multiple parameters such as; polymer purity, bromine and antimony content, as well as presence of rare earth elements (REEs). In this respect DART-HRMS demonstrated an excellent ability to identify BFRs in samples at WEEE relevant concentrations, and in certain cases, volatile antimony species could be detected. DART-HRMS can be used complementary to X-ray fluorescence (XRF) spectroscopy and thermal desorption GC-MS. However, more efforts to characterize DART-HRMS sensitivity limits for antimony detection are needed to ensure DART-HRMS adds value as a stand-alone screening technique for WEEE in contaminated polymers and consumer goods.

• Klíčová slova
• Brominated flame retardants, Direct analysis in real time, High resolution mass spectrometry, Waste electrical and electronic equipment,
• MeSH
• antimon analýza   MeSH
• brom analýza   MeSH
• elektronický odpad analýza   MeSH
• hmotnostní spektrometrie MeSH
• pilotní projekty MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
• polymery chemie   MeSH
• retardanty hoření analýza   MeSH
• spektrometrie rentgenová emisní MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antimon MeSH
• brom MeSH
• polymery MeSH
• retardanty hoření MeSH

Very-long-chain and multibranched polyunsaturated fatty acids of three freshwater sponges, Ephydatia syriaca, Nudospongilla sp., and Cortispongilla barroisi, were studied by silver TLC, GC-MS, UV, IR, HRMS, and NMR methods. One hundred and eighty-five conventional fatty acids were identified by GC-MS, out of which five were new multibranched polyunsaturated fatty acids. The freshwater sponges belonging to the family Spongillidae (class Demospongia) were shown to contain novel di-, tri-, and tetramethyl substituted dienoic, tetraenoic, and hexaenoic fatty acids. The compounds gave positive results in a brine shrimp toxicity assay.

• MeSH
• antibakteriální látky chemie   izolace a purifikace   MeSH
• Artemia účinky léků   MeSH
• Bacillus subtilis účinky léků   MeSH
• chromatografie na tenké vrstvě MeSH
• molekulární struktura MeSH
• nenasycené mastné kyseliny chemie   izolace a purifikace   MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
• Porifera chemie   MeSH
• sladká voda MeSH
• spektrofotometrie infračervená MeSH
• spektrofotometrie ultrafialová MeSH
• Staphylococcus aureus účinky léků   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Geografické názvy
• Izrael MeSH
• Jordánsko MeSH
• Názvy látek
• antibakteriální látky MeSH
• nenasycené mastné kyseliny MeSH

Muscotoxins are cyanobacterial cyclic lipopeptides with potential applications in biomedicine and biotechnology. In this study, Desmonostoc muscorum CCALA125 strain extracts were enriched by polymeric resin treatment, and subjected to HPCCC affording three cyclic lipopeptides (1⁻3), which were further repurified by semi-preparative HPLC, affording 1, 2, and 3, with a purity of 86%, 92%, and 90%, respectively. The chemical identities of 2⁻3 were determined as muscotoxins A and B, respectively, by comparison with previously reported ESI-HRMS/MS data, whereas 1 was determined as a novel muscotoxin variant (muscotoxin C) using NMR and ESI-HRMS/MS data. Owing to the high yield (50 mg), compound 2 was broadly screened for its antimicrobial potential exhibiting a strong antifungal activity against Alternaria alternata, Monographella cucumerina, and Aspergillus fumigatus, with minimum inhibitory concentration (MIC) values of 0.58, 2.34, and 2.34 µg/mL; respectively, and weak antibacterial activity against Bacillus subtilis with a MIC value of 37.5 µg/mL. Compounds 1 and 3 were tested only against the plant pathogenic fungus Sclerotinia sclerotiorum due to their low yield, displaying a moderate antifungal activity. The developed chromatographic method proved to be an efficient tool for obtaining muscotoxins with potent antifungal properties.

• Klíčová slova
• antimicrobial activity, cyanobacteria, cyclic lipopeptides (CLPs), high performance countercurrent chromatography (HPCCC), muscotoxins, γ-methylproline,
• MeSH
• antiinfekční látky chemie   izolace a purifikace   farmakologie   MeSH
• Ascomycota účinky léků   MeSH
• Bacillus subtilis účinky léků   MeSH
• bakteriální toxiny chemie   izolace a purifikace   farmakologie   MeSH
• cyklické peptidy chemie   farmakologie   MeSH
• lipopeptidy chemie   farmakologie   MeSH
• mikrobiální testy citlivosti MeSH
• molekulární struktura MeSH
• sinice metabolismus   MeSH
• syntetické pryskyřice chemie   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antiinfekční látky MeSH
• bakteriální toxiny MeSH
• cyklické peptidy MeSH
• lipopeptidy MeSH
• syntetické pryskyřice MeSH